Viruses are intracellular parasites which require the integrity of certain cell functions so that their replicative cycle within the cells can be successfully performed. Dynein has demonstrated having a relevant role in different steps of virus infection in different viral models such as rabies virus, the human Herpes simplex virus, type I or human immunodeficiency virus. Dynein is a microtubular motor protein, which intervenes in the intracellular transport linked to microtubules and the endosomal pathway and is modulator of different routes of translation of intracellular signals, among other functions. Viruses use dynein for their internalization and intracellular transport, for the formation of the viral factory where the new virions are going to be produced and for the regulation of the cellular signalling necessary in the coordination of these and other processes.
In particular, the protein p54 of the African swine fever virus (ASFV), interacts with a cell protein which is part of the microtubular motor complex called dynein and its function is essentially related to intracellular transport [1]. This interaction was found using the double hybrid system in yeast (an heterologous system), searching for interactor proteins in a swine macrophages cDNA library for possible interaction proteins of the viral p54 protein. The sequence coding for p54 (E183L gene) is included in the complete sequence of the BA71V isolate, deposited in the NCBI database with accession number UI8466. The clones obtained and identified as positive, were sequenced to discover that they contained the complete coding sequence of the light chain dynein of 8 kilodaltons (kDa), called DLC8, LC8, DLC1, DNLC1 or PIN (inhibitor protein of the neuronal nitric oxide synthetase). The sequence coding for DLC8 in Sus scrofa has been deposited in the NCBI database with number AF436777. These results were corroborated using another type of techniques including affinity chromatography, immunoprecipitation and colocalization of both components by confocal microscopy. Those results only confirmed the interaction between p54 protein of ASFV and DLC8.
DLC8 is a protein with a highly conserved nucleotide amino acid sequence between evolutionary distant species (from nematodes to man)[2, 3]. Cytoplasmic dyneins are a family of molecular motors which drive different loads throughout the microtubules. They are in charge of the transport of vesicles, endosomes and organelles from the exterior of the cell to the interior, to the nuclear or perinuclear zone. They are large multiprotein complexes, constituted by one to three heavy chains with a globular head and ATPase activity, which are responsible for generating the necessary energy to produce movement. Bound to these heavy chains are a variable number of intermediate chains and light chains. The latter are responsible for directly interacting with the load to transport. To date, seven families of light chains have been described, among which we can find DLC8. DLC8 is disposed as a dimer in viva, which permits the existence of two identical places of binding of different sequences among both monomers.
With respect to the cellular protein, two types of preferential binding sites with which they interact have been discovered for DLC8 [12, 13]. One of the motifs (Lys/Arg)XThrThr (with X being any amino acid) SEQ ID NO: 15 binds DLC8 with a series of molecules such as the intermediate chain of the dynein, the proapoptotic molecule Bim, Kid1 and Swallow transcription factors and some viral proteins of diverse origin. This binding site is located between the two dimers of the DLC8 molecule. The second motif is: Gly(Ile/Val)GlnValAsp SEQ ID NO: 16 which binds DLC8 to the neuronal nitric oxide synthetase (nNOS) or with the neuronal scaffolding protein, as described so far.
To identify the amino acid residues required for the binding of the viral protein to dynein, several truncated fragments of the p54 protein were expressed and tested in the yeast system to determine that the binding zone to DLC8 is located at the carboxy-terminal end of the p54 protein in 13 amino acids comprised between Tyr149 and Thr161 (TyrThrThrThrValThrThrGlnAsnThrAlaSerGlnThr) SEQ ID NO: 13 [1].
Several viruses use light chain dynein (DLC8) in different stages of their infective cycle inside the host cell. By a technique called pep-scan, peptides mimicking the linear sequences of different proteins of viral origin were synthesized, blotted on to filter paper and probed with DLC8 to determine which linear sequences were suitable for that interaction [10]. The linear sequences that appear below would be theoretically suitable for DLC8 binding. These frequently contain Gln (Q) residues, with often a T residue (Thr) contiguous in the sequence:                The TyrAlaSerGlnThr SEQ ID NO: 17 motif of the p54 protein of the African swine fever virus        The TyrSerThrGlnThr SEQ ID NO: 18 motif of the binding glycoprotein of respiratory syncytial virus        The LysSerThrGlnThr SEQ ID NO: 19 motif of the P protein of the rabies virus and of the Mokola virus, of the helicase of the human herpes simplex virus, of the adenovirus protease, or of the A. moorei entomopoxvirus.        The LysGlnThrGlnThr SEQ ID NO: 20 motif of the E4 protein of the human papillovirus or of the vaccinia virus polymerase.        The LysGlnThrGlnThr SEQ ID NO: 20 motif of gene U19 of the human herpes virus        The ArgValMetGlnLeu SEQ ID NO: 21 motif of the protein of the capsid of human Coxsackievirus, etc.        
Even though some linear viral protein sequences have been found to be theoretically capable to bind DLC8, this does not preclude that all these sequences should be suitable for the interaction in the protein native form or as the molecule is integrated in the motor complex in vivo. Also, it is not demonstrated that those viral sequences are exposed somehow in the viral particle nor their putative binding sites to be able to bind DLC8 in fact, and/or if these viral proteins are synthesized in cellular compartments accessible to dynein during infection such as the cytosol (not endoplasmic reticulum or other secluded organelles and structures). Moreover, none of these linear sequences have been demonstrated to date to be able to block the binding of a given protein to DLC8 by any means, and finally, nothing guarantees that blocking this site would result in infection inhibition. In fact, there are two putative binding sites per DLC8 molecule as above described and there are a number of other light and intermediate chains that could be used alternatively by any given virus. In summary, none of these findings demonstrate that blocking this site would disrupt the interaction nor hamper virus infection and nothing guarantees that above mentioned sequences might be useful as antiviral compounds.
Moreover, for any peptide to be candidate to be used as antiviral, it should reach by some means the intracellular environment adequately and null or very low toxicity in living cells must also be assured. The fact that amino acid sequences may be identified as involved in the binding between viral proteins and DLC8, when those sequences stay on primary (linear) structure does not preclude, that those linear sequences will inhibit viral protein and DLC8 interaction and, accordingly, the peptides comprising those amino acid sequences may serve as antiviral compounds. Both interaction surfaces should be analyzed (for example by their nuclear magnetic resonance spectra) to design a peptide sequence suitable to block the protein-protein interaction. The reason for that lays on the fact that linear amino acid sequences, when folded in higher complexity structure in the cell, as a way of example, bound to a macromolecular complex called the microtubular motor complex, may hind the aminoacid residues involved in the binding with either DLC8 or the viral protein and, therefore, those secondary structure folded peptides would not show any anti-viral activity. Also, a defined sequence that in vitro or in an heterologous system as yeast would be involved in binding might not be exposed in the context of the viral particle and/or might be synthesized in a secluded organelle or structure, making it inaccessible to the cellular protein in the infection in mammalian cells. In all this cases, a peptide theoretically able to block interaction would not show any antiviral activity. An additional reason is that linear peptides when dissolved in the cell cytosol have a tendency to form aggregates which, on turn, would hid again the amino acids responsible of the binding either to DLC8 or to viral proteins. Those aggregated peptides would neither show anti-viral properties. Another important aspect in the design of novel anti-virus peptides relates to their toxicity in non-infected cells. A commercial anti-viral substance should prevent and/or inhibit viral infection but, preferably, without affecting cell viability and cell proliferation of non-infected cells. Last but not least, the invention has found that the amino acids surrounding the motifs involved in DLC8-viral proteins binding area, and particularly their hydrophobicity, they do play an important role in the binding inhibition capabilities of those peptides, to be considered as true anti-viral compounds. Antiviral peptides to achieve an effective inhibition of binding of viral proteins to DLC8 must be fully dissolved into the cell cytosol and, accordingly, the hydrophobicity and proline content of the amino acids neighbouring the binding motifs is crucial.
For all these reasons, it is necessary to generate antiviral strategies to block infection of said viruses by interference with the use that the viruses make of the cell dynein, i.e. either blocking its function or the binding sites which permit the different proteins of viral origin to use the dynein adequately. However, although some overlapping partial amino acid sequences, present in those virus, are repeated as binding motifs to DLC8, as KSTQT SEQ ID NO: 22 or GIQVD SEQ ID NO: 16 , it is also important to inspect the neighbouring residues to assess if the interaction virus-DLC8 will effectively take place. Specific changes on those amino acids were assessed for their abilities to abolish interaction with DLC8.
We have demonstrated using a viral model (African swine fever virus, ASFV), that interfering of the system DLC8-Dynein, entails the blocking of the infection which provides the principal test for a new antiviral strategy which constitutes the object of the present invention.
We have compared the interaction domains of some of the proteins involved in the interaction and its flanking sequences and this information has been used to design peptides which act as the principal antagonists of the interaction of the pair of proteins which interact but that, at the same time, are tagged to reach the intracellular environment adequately and fulfil with all the requirements an antiviral compound must meet, mainly, as previously stated: specific inhibition of viral proteins—DLC8 binding in determined conditions, accessibility and solubility in the cell cytosol, no formation of aggregates and not interference on cell viability and proliferative capacity (no cell toxicity).
In conclusion, the present invention discloses for first time the use as anti-viral compounds of peptides, that were designed based in the sequence (total or partial) by which a virus binds dynein DLC8, as a necessary step for infection success, and those peptides are shown to be efficient inhibiting the viral infection in susceptible cells and to have a demonstrable antiviral effect. The inhibition of virus-DCL8 interaction is reflected in an inhibition of the viral cytopathic effect and a drastic reduction in the number of infected cells. Also, the antiviral effect of this compounds was measured quantitatively to compare their relative efficacy with quantitative PCR in terms of reduction in the copies of viral genomes per cell (which reflects the reduction in the viral replication in ng/μl found in the cell), and also it was measured the consequently significant reduction in virus production and in the synthesis of viral proteins. The invention is exemplified by peptides produced based in the p54 sequence of various ASFV isolates which prevent the infection to progress, being the basis for an antiviral therapy.